Optical scanners find use in performing detection for various experiments, assays and the like. They are often used in array analysis systems for detection of surface bound binding complexes in genomic and proteomic applications, often in connection with microarray devices.
Array use sometimes involves “target” spots of DNA (or RNA) bound to a substrate. Each spot, or sample, of DNA constitutes a separate experiment. To conduct an experiment, “Probe” DNA or RNA which has been labeled with a fluorophor is then introduced to the surface of the slide and is allowed to hybridize with the target DNA. Suitable fluorophores including fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3 (green), Cy3.5, Cy5 (red), Cy5.5, Cy7, FluorX (Amersham) and others, as are well known (see Kricka, 1992, Nonisotopic DNA Probe Techniques, Academic Press San Diego, Calif.). Excess probe DNA that does not bind with target DNA is removed from the surface of the slide in a subsequent washing process.
Such an experiment allows the binding affinity between the probe and target DNA to be measured to determine the likeness of their molecular structures; complementary molecules have a much greater probability of binding than unrelated molecules. The probe DNA is labeled with fluorescent labels that emit a range of radiation energy centered about and including a certain wavelength when excited by an external radiation source of a shorter wavelength. The brightness of emitted radiation is a function of the fluor density in the illuminated sample. Because the fluor density is a function of the binding affinity or likeness of the probe molecule to the target molecule, the brightness of each sample can be mapped as to the degree of similarity between the probe DNA and the target DNA present. On a typical microarray, up to tens of thousands of experiments can be performed simultaneously on the probe DNA, allowing for a detailed characterization of complex molecules.
An optical scanner is used to retrieve such data as is available. Generally, fluorescence-based micro-array scanners incorporate the ability to deliver multiple laser excitation wavelengths so that fluorescence data can be obtained from the sample at two or more emission wavelengths by detecting two or more fluorescent dyes. Many DNA micro-arrays are utilized in connection with a two-wavelength scanning method, where the results of one wavelength scan are used as control values and the results of the other wavelength scan represent the desired experimental result. Such an approach is employed in Differential Gene Expression assays.
Typically, pairs (or increasing numbers) of fluorophores are chosen that have distinct emission spectra so that they can be easily distinguished by the scanner. However, as the market and applications mature, and a larger variety of suitable dyes become available, the demand for alternative excitation wavelengths and emission bands will increase. U.S. Pat. No. 6,355,934 is directed to systems suited to accommodate such future needs, thereby providing background for potential applications of the present invention.
Whatever the case, the present invention involves approaches for achieving full data acquisition. In order to properly interpret data obtained from a plurality of scanned channels, it has been appreciated that the intensity of the recorded signal for the different channels should be averaged or normalized. Otherwise, as in Differential Gene Expression tests, sample data and control data will be skewed relative to each other. It has been appreciated that system asymmetries can produce unbalanced results between channels that should otherwise produce an equivalent intensity based on some metric.
To account for this, a number of approaches have been developed by which normalization or channel balance may be attempted. One approach described in the aforementioned U.S. Pat. No. 6,351,712 is to set average signal intensity for the scanned channels to an equivalent value after the data has been obtained, thereby scaling the other results obtained. Another procedure described therein involves normalization between channels using the intensity ratio of genomic DNA spots.
U.S. Pat. No. 6,344,316 describes another software or post-data acquisition processing approach. Here, a system is described in which the signal intensities obtained from scanning oligonucleotide control probes that are perfectly complementary to a labeled reference are used as a mathematical devisor for feature intensity readings taken by each corresponding channel.
In contrast, U.S. Pat. No. 6,078,390 describes a system which operates by performing a partial scan and then adjusting each channel so that the brightest detected features are, on average, set to a predetermined maximum unsaturated value. This system implicitly relies on the assumption that a full scan of an array will yield results consistent with the partial scan results.
Each of these approaches has its drawbacks. The image post-processing techniques involving software manipulation of data that can result in loss or clipping of high-end or low-end data which is not acquired due to setup inadequacy (i.e., non-optimal detector gain or excitation source power settings). The system described in the '390 patent also relies on assumptions that may result in failure to acquire what data is available. Furthermore, by balancing channels in view of the most intense readings obtained, the likelihood of error is increased since maximum intensity features will generally be the least common signal received.
Another disadvantage of what is taught by the '390 patent is that the brightest red and green features will not necessarily have a known ratio. When running an experiment with a two-color array, the differential expression of the sample on various probe features is what is being measured. Therefore, the brightest features of red and green may not have a ratio equal to or substantially equal to 1 even while the ratio of the average red and average green features is equal to 1. As such, the channels may actually be set out of balance by the regime taught in that patent. This will skew scanner results rather than appropriately normalize them.
Accordingly, there exists a need for a scanner system more closely tied to the details and/or realities of data acquisition in which potential data is not lost, and in which reliable data—not merely prospectively representative data—is used to balance or equalize the experimental channels using adjustments in the scanner. The present invention meets this need. Other needs and advantages presented by the present invention may also be apparent to those with skill in the art upon review of the following disclosure.